Fluorescent reporters of thrombin, heparin cofactor II, and heparin binding in a ternary complex.
Fluorescent reporters of thrombin, heparin cofactor II, and heparin binding in a ternary complex.
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凝血酶、肝素辅因子 II 和肝素结合在三元复合物中的荧光报告基因。
DOI：
10.1016/j.ab.2011.11.021
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发表时间：
2012
影响因子：
2.9
通讯作者：
Verhamme,IngridM
中科院分区：
文献类型：
--
作者：
Verhamme,IngridM
肝素辅助因子II （HCII）的凝血酶失活通过与肝素形成三元络合物而加速。新的活性位点标记的凝血酶[4'F]FPR-T和[6F]FFR-T，以及外源位点I探针Hir-(54-65)(SO3-)，表征了凝血酶外源位点I和II与复合物中HCII和肝素的相互作用。HCII结合肝素结合[4'F]FPR-T的外源位点I引起饱和荧光增加，抗凝血酶不存在。肝素与外源位点II和第二个较弱位点的结合导致[6F]- ffr - t荧光猝灭，并通过Hir-(54-65)（SO3-）同时结合而减弱。停流分析显示HCII和[6F]FFR-T·肝素复合物有序组装，这与肝素与凝血酶的结合比与HCII的结合更紧密一致。饱和HCII依赖性和钟形肝素依赖性的荧光变化报道了三络合物的形成，这与凝血酶·肝素复合物结合HCII并允许凝血酶·（肝素）2复合物与HCII相互作用的模板机制一致。Hir-(54-65)（SO3-）置换与fpr阻断凝血酶和活性凝血酶反应表明，在三元配合物形成过程中，活性位点和外源位点I协同作用。这些研究表明，与单独的肝素结合相比，HCII与凝血酶·肝素复合物的结合显著增强，并且当凝血酶上的肝素结合位点都饱和时，外源位点I仍然可用于配体或HCII结合。
Thrombin inactivation by heparin cofactor II (HCII) is accelerated by ternary complex formation with heparin. The novel active-site-labeled thrombins, [4′F]FPR-T and [6F]FFR-T, and the exosite I probe, Hir-(54–65)(SO3-), characterized thrombin exosite I and II interactions with HCII and heparin in the complex. HCII binding to exosite I of heparin-bound [4′F]FPR-T caused a saturable fluorescence increase, absent with antithrombin. Heparin binding to exosite II and a second weaker site caused fluorescence quenching of [6F]-FFR-T, attenuated by simultaneous Hir-(54–65)(SO3-) binding. Stopped-flow analysis demonstrated ordered assembly of HCII and the [6F]FFR-T·heparin complex, in agreement with tighter heparin binding to thrombin than to HCII. Saturating HCII dependences and bell-shaped heparin dependences of the fluorescence change reported ternary complex formation, consistent with a template mechanism in which the thrombin·heparin complex binds HCII and allowing for interaction of thrombin·(heparin)2complexes with HCII. Hir-(54–65)(SO3-) displacement in reactions with FPR-blocked and active thrombin indicated a concerted action of the active site and exosite I during ternary complex formation. These studies demonstrate that binding of HCII to the thrombin·heparin complex is dramatically enhanced compared with heparin binding alone and that exosite I is still available for ligand or HCII binding when both heparin binding sites on thrombin are saturated.